The porphyrins and related tetrapyrrole macrocycles are among the most versatile of tetradentate ligands.sup.1. Attempts to stabilize higher coordination geometries with larger porphyrin-like aromatic macrocycles have met with little success..sup.2-13 Only the uranyl complex of "superphthalocyanine" has been isolated and characterized structurally,.sup.2 although several other large porphyrin-like aromatic macrocycles, including the "sapphyrins",.sup.3,6 "oxosapphyrins",.sup.6,7 "platyrins",.sup.8 "pentaphyrin",.sup.9 and "[26]porphyrin",.sup.10 have been prepared in their metal free forms. Large, or "expanded" porphyrin-like systems are of interest for several reasons: They could serve as aromatic analoguss of the better studied porphyrins.sup.2-10 or serve as biomimetic models for these or other naturally occurring pyrrole-containing systems..sup.36,13a In addition, large pyrrole containing systems offer possibilities as novel metal binding macrocycles..sup.2,4,5,13b,35,14 For instance, suitably designed systems could act as versatile ligands capable of binding larger metal cations and/or stabilizing higher coordination geometries.sup.2 than those routinely accommodated within the normally tetradentate ca. 2.0 .ANG. radius porphyrin core..sup.21 The resulting complexes could have important application in the area of heavy metal chelation therapy, serve as contrast agents for magnetic resonance imaging (MRI) applications, act as vehicles for radioimmunological labeling work, or serve as new systems for extending the range and scope of coordination chemistry..sup.14,39 In addition, the free-base (metal-free) and/or diamagnetic metal-containing materials could serve as useful photosensitizers for photodynamic therapeutic applications. In recent years a number of pentadentate polypyrrolic aromatic systems, including the "sapphyrins",.sup.3,6 "oxosapphyrins",.sup.7 "smaragdyrins",.sup.3,6 "platyrins",.sup.8 and "pentaphyrin".sup.9 have been prepared and studied as their metal-free forms. For the most part, however, little or no information is available for the corresponding metallated forms. Prior to this invention the uranyl complex of "superphthalocyanine" was the only metal-containing pentapyrrolic system which has been prepared and characterized structurally..sup.2 The "superphthalocyanine" system is not capable of existence in either its free-base or other metal-containing forms..sup.2 Thus, prior to the present invention, no versatile, structurally characterized, pentadentate aromatic ligands were available,.sup.13b although a number of nonaromatic pyridine-derived pentadentate systems had previously been reported..sup.37,38
Gadolinium(III) complexes derived from strongly binding anionic ligands, such as diethylenetriamine pentaacetic acid (DTPA),.sup.40,42 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA),.sup.40,43,44 and 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane-N,N'-diacetic acid (dacda),.sup.40,45 are among the most promising of the paramagnetic contrast agents currently being developed for use in magnetic resonance imaging (MRI).sup.40 The complex, [Gd.cndot.DTPA]-, is now being used clinically in the United States in certain enhanced tumor detection and other imaging protocols..sup.40 Nonetheless, the synthesis of other gadolinium(III) complexes remains of interest since such systems might have greater kinetic stability, superior relaxivity, or better biodistribution properties than this or other carboxylate-based contrast agents. The water-soluble porphyrin derivatives, such as tetrakis(4-sulfonatophenyl)porphyrin (TPPS) cannot accommodate completely the large gadolinium(III) cation.sup.47 within the relatively small porphyrin binding core (r.congruent.2.0 .ANG..sup.48), and, as a consequence, gadolinium porphyrin complexes are invariably hydrolyrically unstable..sup.33,34,46,49,50 Larger porphyrin-like ligands may offer a means of circumventing this problem..sup.51,59
A promising new modality for use in the control and treatment of tumors is photodynamic therapy (PDT)..sup.60-64 This technique uses of a photosensitizing dye, which localizes at, or near, the tumor site, and when irradiated in the presence of oxygen serves to produce cytotoxic materials, such as singlet oxygen (O.sub.2 (.sup.1 .DELTA..sub.g)) from benign precursors (e.g. (0.sub.2 (.sup.3 .SIGMA..sub.g -)). Diamagnetic porphyrins and their derivatives are the dyes of choice for PDT. It has been known for decades that porphyrins, such as hematoporphyrin, localize selectively in rapidly growing tissues including sarcomas and carcinomas..sup.65 The hematoporphyrin derivative (HPD),.sup.61-64,66-80 is an incompletely characterized mixture of monomeric and oligomeric porphyrins..sup.81-86 The oligomeric species, which are believed to have the best tumor-localizing ability,.sup.82,85 are marketed under the trade name Photofrin II.RTM. (PII) and are currently undergoing phase III clinical trials for obstructed endobronchial tumors and superficial bladder tumors. The mechanism of action is thought to be the photoproduction of singlet oxygen (O.sub.2 (.sup.1 .DELTA..sub.g)), although involvement of superoxide anion or hydroxyl and/or porphyrin-based radicals cannot be entirely ruled out..sup.87,92 Promising as HPD is, it and Other available photosensitizers (e.g., the phthalocyanines and naphthalocyanines) suffer from serious disadvantages.
While porphyrin derivatives have high triplet yields and long triplet lifetimes (and consequently transfer excitation energy efficiently to triplet oxygen),.sup.101b,g their absorption in the Q-band region parallels that of heme-containing tissues. Phthalocyanines and naphthalocyanines absorb in a more convenient spectral range but have significantly lower triplet yields;.sup.102 moreover, they tend to be quite insoluble in polar protic solvents, and are difficult to functionalize. Thus the development of more effective photochemotherapeutic agents requires the synthesis of compounds which absorb in the spectral region where living tissues are relatively transparent (i.e., 700-1000 nm),.sup.99d have high triplet quantum yields, and are minimally toxic. The present inventors have recently reported.sup.103 (see Example 1) the synthesis of a new class of aromatic porphyrin-like macrocycles, the tripyrroledimethine-derived "texaphyrins", which absorb strongly in the tissue-transparent 730-770 nm range. The photophysical properties of metallotexaphyrins parallel those of the corresponding metalloporphyrins and the diamagnetic complexes sensitize the production of .sup.1 O.sub.2 in high quantum yield.
Acquired immunodeficiency syndrome (AIDS) is among the most serious public health problems facing our nation today. AIDS, first reported in 1981 as occurring among male homosexuals,.sup.60 is a fatal human disease which has now reached pandemic proportions. At present, sexual relations and needle-sharing are the dominant mechanisms for the spread of AIDS..sup.60 Since the testing of blood supplies began, the percentage of AIDS infections due to blood transfusions has dropped considerably..sup.60,104-107 However, an absolutely fail-proof means must be developed to insure that all stored blood samples are free of the AIDS virus (and ideally all other blood-borne pathogens). Serologic tests for HIV-1 are insufficient to detect all infected blood samples, in particular, those derived from donors who have contracted the disease but not yet produced detectable antibodies..sup.104-107
Any blood purification procedure used to remove AIDS virus or other blood-borne pathogens should operate without introducing undesirable toxins, damaging normal blood components, or inducing the formation of harmful metabolites. This precludes the use of common antiviral systems such as those based on heating, UV irradiation, or purely chemical means. A promising approach is the photodynamic one alluded to above. Here, preliminary studies, carried out by researchers at the Baylor Research Foundation, Dr. Matthews and his team,.sup.93-96 and others,.sup.97,98 have served to show that HPD and PII, in far lower dosages than are required for tumor treatment, act as efficient photosensitizers for the photo-deactivation of cell-free HIV-1, herpes simplex (HSV), hepatitis and other enveloped viruses. The success of this procedure derives from the fact that these dyes localize selectively at or near the morphologically characteristic, and physiologically essential, viral membrane ("envelope") and catalyze the formation of singlet oxygen upon photoirradiation. The singlet oxygen destroys the essential membrane envelope. This kills the virus and eliminates infectivity. Photodynamic blood purification procedures, therefore, rely on the use of photosensitizers which localize selectively at viral membranes, just as more classic tumor treatments require dyes that are absorbed or retained preferentially at tumor sites. Simple enveloped DNA viruses like HSV-1 are good models for testing putative photosensitizers for potential use in killing the far more hazardous HIV-1 retrovirus. This correspondence holds only as far as freely circulating (as opposed to intracellular) viruses are concerned. Complete prophylactic removal of HIV-1 from blood products will require the destructive removal of the virus from within monocytes and T lymphocytes..sup.108
This "first generation" of dyes suffers from a number of serious deficiencies which may militate against their eventual use in biomedical applications. Each of these deficiencies has important clinical consequences. Since HPD and PII do not contain a single chemically well-defined constituent, coupled with the fact that the active components have yet to be identified with certainty,.sup.82-86 means that the effective concentrations vary from preparation to preparation. Thus the dosage, and the light fluence, cannot be optimized and predetermined for any particular application. Since they are not metabolized rapidly, significant quantities of these dyes remain in stored blood units after prophylactic photoinduced HIV-1 removal and remain in patients' bodies long after photodynamic tumor treatment. The latter retention problem, in particular, is known to be serious; HPD and PII localize in the skin and induce photosensitivity in patients for weeks after administration..sup.64,109 Since the longest wavelength absorption maximum for these dyes falls at 630 nm, most of the incipient energy used in phototreatment is dispersed or attenuated before reaching the center of a deep-seated tumor and as a result, little of the initial light is available for singlet oxygen production and therapy..sup.110-112 A study using a mouse model with a 3 mm tumor implanted beneath the skin indicated that as much as 90% of the energy is lost by the base of the tumor..sup.110 More effective treatment of deep-seated or large tumors may be possible if photosensitizers could be developed which absorb in the &gt;700 nm region, provided, of course, they retain the desirable features of HPD and PII (e.g. selective localization in target tissues and low dark toxicity). One aspect of the present invention involves development of such improved photosensitizers for use in photodynamic tumor treatment and blood purification protocols.
The following list summarizes features which would be desirable in biomedical photosensitizers:
1. Easily available PA1 2. Low intrinsic toxicity PA1 3. Long wavelength absorption PA1 4. Efficient photosensitizer for singlet oxygen production PA1 5. Fair solubility in water PA1 6. Selective up-take in tumor tissue and/or PA1 7. Showing high affinity for enveloped viruses PA1 8. Quick degradation and/or elimination after use PA1 9. Chemically pure and stable PA1 10. Easily subject to synthetic modification PA1 1) appreciable solubility, particularly in aqueous media; PA1 2) biolocalization in desired target tissue; PA1 3) the ability to attach to solid matrices; PA1 4) the ability to be attached to biomolecules; PA1 5) efficient chelation of divalent and trivalent metal cations; PA1 6) absorption of light in the physiologically important PA1 region of 690-880; PA1 7) high chemical stability; PA1 8) ability to stabilize diamagnetic complexes that form long-lived triplet states in high yield and that act as efficient photosensitizers for the formation of singlet oxygen. PA1 wherein the substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently hydrogen, [H]; PA1 hydroxyl, [OH]; PA1 alkyl groups attached via a carbon or oxygen; PA1 hydroxyalkyl groups attached via a carbon or oxygen; these may be C.sub.n H.sub.(2n+1) O.sub.y or OC.sub.n H.sub.(2n+1) O.sub.y ; where at least one of the subtituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 has at least one hydroxy substituent; where the molecular weight of any one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, or R.sub.5 is less than or equal to about 1000 daltons; where n is a positive integer or zero; and PA1 where y is zero or a positive integer less than or equal to (2n+1); PA1 oxyhydroxyalkyl groups (containing independently hydroxy substituents or ether branches) attached via a carbon or oxygen; these may be C.sub.(n-x) H.sub.[(2n+1)-2x] O.sub.x O.sub.y or OC.sub.(n-x) H.sub.[(2n+1)-2x] O.sub.x O.sub.y ; where n is a positive integer or zero, x is zero or a positive integer less than or equal to n, and y is zero or a positive integer less than or equal to [(2n+1)-2x]; PA1 oxyhydroxyalkyl groups (containing independently substituents on the hydroxyls of the oxyhydroxyalkyl compounds described above or carboxyl derivatives) attached via a carbon or oxygen; these may be C.sub.n H.sub.[(2n+1)-q] O.sub.y R.sup.a.sub.q, OC.sub.n H.sub.[(2n+1)-q] O.sub.y R.sup.a q or (CH.sub.2).sub.n CO.sub.2 R.sup.a ; where n is a positive integer or zero, y is zero or a positive integer less than [(2n+1)-q], q is zero or a positive integer less than or equal to 2n+1, R.sup.a is independently H, alkyl, hydroxyalkyl, saccharide, C.sub.(m-w) H.sub.[(2m+1)-w] O.sub.w O.sub.z, O.sub.2 CC.sub.(m-w) H.sub.[(2m+1)-2w] O.sub.w O.sub.z or N(R)OCC.sub.(m-w) H[(2M-30 1)-2w]O.sub.w O.sub.z ; where m is a positive integer or zero, w is zero or a positive integer less than or equal to m, z is zero or a positive integer less than or equal to [(2m+ 1)-2w], R is H, alkyl, hydroxyalkyl, or C.sub.m H.sub.[(2m+1)-r] O.sub.z R.sup.b .sub.r ; where m is a positive integer or zero, z is zero or a positive integer less than [(2m+1)-r], r is zero or a positive integer less than or equal to 2m+1, and R.sup.b is independently H, alkyl, hydroxyalkyl, or saccharide; PA1 carboxyamidealkyl groups (containing independently hydroxyl groups, or secondary or tertiary amide linkages) attached via a carbon or oxygen; these may be ( CH.sub.2 ).sub.n CONHR.sup.a, O(CH.sub.2).sub.n CONHR.sup.a, (CH.sub.2).sub.n CON(R.sup.a).sub.2, or O(CH.sub.2).sub.n CON(R.sup.a).sub.2 ; where n is a positive integer or zero, R.sup.a is independently H, alkyl, hydroxyalkyl, saccharide, C.sub.(m-w) H.sub.[(2m+1)-2w] O.sub.w O.sub.z, O.sub.2 CC.sub.(m-w) H.sub.[(2m+1)-2w] O.sub.w O.sub.z or N(R)OCC.sub.(m-w) H.sub.[(2m+1)-2 w]O.sub.w O.sub.z ; where m is a positive integer or zero, w is zero or a positive integer less than or equal to m, z is zero or a positive integer less than or equal to [(2m+1)-2w], R is H, alkyl, hydroxyalkyl, or C.sub.m H.sub.[(2m+1)-r] O.sub.z R.sup.b.sub.r ; where m is a positiveinteger or zero, z is zero or a positive integer less than [(2m+1)-r], r is zero or a positive integer less than or equal to 2m+1, and R.sup.b is independently H, alkyl, hydroxyalkyl, or saccharide; or PA1 carboxyalkyl groups (containing independently hydroxyl groups, carboxyl substituted ethers, amide substituted ethers or tertiary amides removed from the ether) attached via a carbon or oxygen; these may be C.sub.n H.sub.[(2n+1)-q] O.sub.y R.sup.c.sub.q or OC.sub.n H.sub.[(2n+1)-q] O.sub.y R.sup.c.sub.q ; where n is a positive integer or zero, y is zero or a positive integer less than [(2n+1)-q], q is zero or a positive integer less than or equal to 2n+1, R.sup.c is (CH.sub.2).sub.n CO.sub.2 R.sup.d, (CH.sub.2).sub.n CONHR.sup.d or (CH.sub.2).sub.n CON(R.sup.d).sub.2 ; where n is a positive integer or zero, R.sup.d is independently H, alkyl, hydroxyalkyl, saccharide, C.sub.(m-w) H.sub.[(2m+1)-2w] O.sub.w O.sub.z, O.sub.2 CC.sub.(m-w)H .sub.[(2m+1)-2w] O.sub.w O.sub.z or N(R)OCC.sub.(m-w) H.sub.[(2m+1)- 2w]O.sub.w O.sub.z ; where m is a positive integer or zero, w is zero or a positive integer less than or equal to m, z is zero or a positive integer less than or equal to [(2m+1)-2w], R is H, alkyl, hydroxyalkyl, or C.sub.m H.sub.[(2m+1)-r] O.sub.z R.sup.b.sub.r ; where m is a positive integer or zero, z is zero or a positive integer less than [(2m+1)-r], r is zero or a positive integer less than or equal to 2m+1, and. R.sup.b is independently H, alkyl, hydroxyalkyl, or saccharide; where at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 has at least one hydroxy substituent and the molecular weight of any of R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5 is less than or equal to about 1000 daltons.
In recent years, considerable effort has been devoted to the synthesis and study of new photosensitizers which might meet these desiderata. Although a few of these have consisted of classic dyes such as those of the rhodamine and cyanine classes,.sup.113,115 many have been porphyrin derivatives with extended .pi. networks..sup.116,126 Included in this latter category are the purpurins and verdins.sup.116 of Morgan and other chlorophyll-like species,.sup.117-119 the benz-fused porphyrins of Dolphin et al ,.sup.120 and the sulfonated phthalocyanines and napthophthalocyanines studied by Ben-Hur,.sup.121 Rodgers,.sup.122 and others..sup.123-127 Of these, only the napthophthalocyanines absorb efficiently in the most desirable &gt;700 nm spectral region. These particular dyes are difficult to prepare in a chemically pure, water soluble form and are relatively inefficient photosensitizers for singlet oxygen production, perhaps even acting photodynamically via other oxygen derived toxins (e.g. superoxide). Thus a search continues for yet a "third generation" of photosensitizers which might better meet the ten critical criteria listed above.
It is an important aspect of the present invention that an improved "third generation" of photosensitizers is obtained using large, pyrrole-containing "expanded porphyrins". These systems, being completely synthetic, can be tuned so as to incorporate any desired properties. In marked contrast to the literature of the porphyrins, and related tetrapyrrolic systems (e.g. phthalocyanines, chlorins, etc.), there are only a few reports of larger pyrrole-containing systems, and only a few of these meet the criterion of aromaticity deemed essential for long-wavelength absorption and singlet oxygen photosensitization..sup.128 In addition to the present inventors' studies of texaphyrin 1.sub.B,.sup.129 (see FIGS. 1B and 2B), and "sapphyrin", first produced by the groups of Woodward.sup.3 and Johnson.sup.6, there appear to be only three large porphyrin-like systems which might have utility as photosensitizers These are the "platyrins" of LeGoff.sup.8, the stretched porphycenes of Vogel.sup.131a and the vinylogous porphyrins of Franck..sup.130 The present studies indicate that an expanded porphyrin approach to photodynamic therapy is promising. The porphycenes,.sup.131b, 131c a novel class of "contracted porphyrins" also show promise as potential photosensitizers..sup.132
The present invention involves a major breakthrough in the area of ligand design and synthesis. It involves the synthesis of the first rationally designed aromatic pentadentate macrocyclic ligand, the tripyrroledimethine-derived "expanded porphyrin" 1.sub.B..sup.129 This compound, to which the trivial name "texaphyrin" has been assigned, is capable of existing in both its free-base form and of supporting the formation of hydrolytically stable 1:1 complexes with a variety of metal cations, such as Cd.sup.2+, Hg.sup.2+, In.sup.3+, Y.sup.3+, Nd.sup.3+, Eu.sup.3+, Sm.sup.3+, La.sup.3+, Lu.sup.3+, Gd.sup.3+, and other cations of the lanthanide series that are too large to be accommodated in a stable fashion within the 20% smaller tetradentate binding core of the well-studied porphyrins. In addition, since the free-base form of 1.sub.B is a monoanionic ligand, the texaphyrin complexes formed from divalent and trivalent metal cations remain positively charged at neutral pH. As a result, many of these complexes are more water soluble than the analogous porphyrin complexes.
To date, two X-ray crystal structures of two different Cd.sup.2+ adducts have been obtained, one of the coordinatively saturated, pentagonal bipyramidal bispyridine complex;.sup.129a the other of a coordinatively unsaturated pentagonal pyramidal benzimidazole complex..sup.129b Both confirm the planar pentadentate structure of this new ligand system and support the assignment of this prototypical "expanded porphyrin" as aromatic.
Further support for the aromatic formulation comes from the optical properties of 1.sub.B and 1.sub.C. The lowest energy Q-type band of the structurally characterized bispyridine cadmium(II) adduct of complex 1.sub.C at 767 nm (.epsilon.=51,900) in CHCl.sub.3 is 10-fold more intense and red shifted by almost 200 nm as compared to that of a typical reference cadmium(II) porphyrin. Compound 1.sub.B and both its zinc(II) and cadmium(II) complexes are very effective photosensitizers for singlet oxygen, giving quantum yields for .sup.1 O.sub.2 formation of between 60 and 70% when irradiated at 354 nm in air-saturated methanol..sup.129c Related congeneric texaphyrin systems bearing substituents on the tripyrrole and/or phenyl portions and incorporating La(III) and/or Lu(III) metal centers, have been found to produce .sup.1 O.sub.2 in quantum yields exceeding 70% when irradiated under similar conditions. Thus, it is this remarkable combination of light absorbing and .sup.1 O.sub.2 photo-sensitizing properties which make these systems ideal candidates for use in photodynamic therapy and blood purification protocols.